JPH0371439B2 - - Google Patents

Abstract

Nonapeptide and decapeptide analogs of LH-RH of the formula (pyro)Glu-His-V-Ser-W-X-Y-Arg-Pro-Z (I) and the pharmaceutically acceptable salts thereof wherein: V is tryptophyl, phenylalanyl or 3-(1-naphthyl)-L-alanyl; W is tyrosyl, phenylalanyl or 3-(1-pentafluorophenyl)-L-alanyl; X is a D-amino acid residue <IMAGE> wherein R is (a) a carbocyclic aryl-containing radical selected from the group consisting of naphthyl, anthryl, fluorenyl, phenanthryl, biphenylyl, benzhydryl and phenyl substituted with three or more straight chain lower alkyl groups; or (b) a saturated carbocyclic radical selected from the group consisting of cyclohexyl substituted with three or more straight chain lower alkyl groups, perhydronaphthyl, perhydrobiphenylyl, perhydro-2,2-diphenylmethyl and adamantyl; Y is leucyl, isoleucyl, nor-leucyl or N-methyl-leucyl; Z is glycinamide or -NH-R1, wherein R1 is lower alkyl, cycloalkyl, fluoro lower alkyl or <IMAGE> wherein R2 is hydrogen or lower alkyl, are disclosed. These compounds exhibit potent LH-RH agonist properties.

Description

DETAILED DESCRIPTION OF THE INVENTION Luteinizing hormone (LH) and follicle stimulating hormone (FSH) are released from the anterior pituitary gland under the control of luteinizing hormone-releasing hormone LH-RH produced in the hypothalamus. LH and FSH act on the reproductive rays to stimulate the synthesis of steroid hormones and the maturation of gametes. The reproductive cycle of livestock and humans is controlled by the pulsatile release of LH-RH and the associated release of LH and FSH. Additionally, LH-RH acts on the placenta, on the release of human chorionic gonadotropin (HCG), and directly on the genital tract. The active homologues of LH-RH are 2
It is useful in controlling pregnancy rates through two mechanisms of action. Small doses of LH-RH congeners stimulate ovulation and are useful in the treatment of hypothalamic and ovulatory infertility. Additionally, it can be used to treat hypogonadal conditions and impotence and to stimulate spermatogenesis and androgen production in males. Conversely, administration of relatively large doses of highly potent and long-lasting LH-RH congeners has the opposite effect, preventing ovulation in females and regulating spermatogenesis in males. including decreased weight of accessory organs in males and females;
A reduction in the normal circulating amount of sex steroids from the genital rays is also associated with the above-mentioned effects. This adverse effect promotes weight gain in livestock when feed is sufficient, makes pregnant animals more likely to miscarry, and generally acts as a chemical sterilizer. The natural hormone-releasing hormone LH-RH is a decapeptide consisting of natural amino acids (excluding the achiral amino acid glycine, which exhibits the L configuration). The arrangement is as follows. (Pyro)Glu-His-
Trp−Ser−Tyr−Gly−Leu−Arg−Pro−Gly−
_NH2 . Although many congeners of this natural substance have been the subject of research, the majority of them have been found to have low biological activity and are not suitable for clinical use. Certain selected species have been found to have beneficial effects on biological activity. A particularly interesting embodiment is obtained by replacing residue 6 from Gly to a D -amino acid. For example, replacing the Gly residue at position 6 with D -Ala, D-
When substituted with Leu, D -Phe or D -Trp, LH-
A series of LH-RH homologues with increased activity compared to RH is obtained. [D-Ala ⁶ ] M. Monahan for LHRH.
et al., Biochem . , 12 , 4616 (1973); [D-Leu ⁶ ]
LHRH and des Gly ¹⁰ [D-Leu ⁶ , Pro NHEt ⁹ ]
For LHRH, JAVilchez−Martinez, et al.
al, Biochem.Biophys.Res.Comm. , 59 , 1226
(1974); [D-Phe ⁶ ] DH for LHRH
Coy, et al, J.Med.Chem. , 19 , 423 (1976); and for [D-Trp ⁶ ]LHRH, W. Vale, et al.
al, Clinical Endocrinology, 5th Supp.
Blackuwell Scientific Publications, Oxford
England (1976), P.2615 and DHCoy, et al;
Biochem.Biophys.Res.Comm., 67 , 576 (1979)
are listed respectively. As mentioned above, substitution at position 6 not only substantially increases activity, but also eliminates Gly- _NH2 at position 10 to provide alkyl-, cycloalkyl- or fluoroalkyl-amide forms. by making it a nonapeptide, or
Activity can be increased by replacing Gly- _NH2 with α-azaglycinamide. For example, M.Fujino, et al, Biochem.Biophys.Res.
Comm., 49 , 863 (1972), DHCoy, et al.
Biochem., 14 , 1848 (1975) and ASDutta, et al.
al, J.Chem.Soc.Perkin I, 1979 . See 379. Substitution of the leucine residue at position 7 with N-methyl-leucine increases safety against oxygen degradation.
For example, N. Ling, et al, Biochem Biophys.
See Res.Comm., 63 , 801 (1975). Substitution of the tryptophan residue at position 3 with 3-(1-naphthyl) -L -alanine increases biological efficacy. For example, KUPrasad, et al, J.Med.
Chem., 19 , 492 (1976) and Y. Yabe, Chem.
See Pharm. Bull., 24 (12), 3149 (1976). The tyrosine residue at position 5 can be replaced with phenylalanine or 3-(1) while retaining substantial biological activity.
-pentafluorophenyl) -L -alanine. For example, N. Yanaihara, et al.
al, Biochem.Biophys.Res.Comm. , 52 , 64
(1973), and D. Coy, et al, J. Med. Chem. , 16 ,
See 877 (1973). Other LH-RH analogues are desired which have higher biological activity than that of LH-RH mentioned above and which can be used clinically in animals and humans. The present invention relates to a method for producing novel peptide derivatives of LH-RH having certain lipophilic D-amino acids in the 6-position. More specifically, the present invention provides a method of formula ()(pyro)Glu-His-Trp by peptide solution synthesis method.
The present invention relates to a method for producing a compound represented by -Ser-Tyr-3-(2-naphthyl)-D-alanyl-Leu-Arg-Pro-Gly- _NH2 () and a pharmaceutically acceptable salt thereof. As mentioned above for convenience in describing the present invention, Biochemistry , 11 , 1726 (1972)
We use the commonly accepted abbreviations for the various amino acids in the peptide art as recommended by the IUPAC-IUB Commission as described in . However, excluding the achiral amino acid glycine, L
- represents an amino acid. All peptide sequences herein are presented in accordance with generally accepted conventions with the N-terminal amino acid written on the left hand side and the C-terminal amino acid written on the right hand side. As used herein, the term "pharmaceutically acceptable salt" refers to salts that maintain the desired biological activity of the parent compound and do not exhibit undesired toxic effects. Examples of such salts include (a) acid addition salts formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid; organic acids such as acetic acid, tartaric acid, succinic acid, maleic acid, Fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid,
Salts formed from naphthalenedisulfonic acid, polygalactronic acid; (b) polyvalent metal cations, e.g.
Salts of zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or salts of organic cations formed from N,N'-dibenzylethylene-diamine or ethylenediamine; or (c) the above. Combinations of (a) and (b), such as zinc tannate salts. The compounds of the present invention, especially the salts, are said to exhibit the greatest LH-RH agonist activity to date (pyro)Glu-His-Trp-Ser-Tyr- D-
It exhibits surprisingly greater and longer lasting LH-RH agonist activity than Trp-Ser-Arg-Pro-Gly- _NH2 and its corresponding prolylethylamide. The first measure of efficacy is the ability to partially or completely suppress oestrus (measured over a period of 3 weeks) observed by subcutaneous injection twice daily in normally cycling adult female rats. Other biological tests performed on LH-RH congeners and compounds of the invention are as follows. (a) Ovulation induction by subcutaneous injection in female rats in diestrus or proestrus (Rippel, et al.
Proc.Soc.Exp.Biol.Med., 148 , 1193 (1975)), (b) Measurement of LH and FSH release by dispersed anterior pituitary cell cultures by radioimmunoassay (Vale, et al, Endocrinology , 91 ,562
(1972)), and (c) the release of LH and FSH into the peripheral circulation observed during intravenous injection into ovarian-deleted steroid-treated rats was measured by radioimmunoassay (Arimura, et al., Endocrinology , 90 , 163
(1972)) At a more advanced level, the activity of these compounds may be associated with the large reduction in prostate size, reduced spermatogenesis and testosterone levels in the circulatory system and testicles seen in dogs with benign prostatic hyperplasia. This can be explained by in-vivo testing due to a decrease in Therefore, the compounds of the present invention can be used in a wide range of situations where regulation of LH and FSH or direct reproductive effects are important. Their uses include: Physiological uses (low dose effects) Ovulation induction and regulation in anovulatory infertility in female mammals, treatment of infertility caused by female luteal dysfunction, reduction of reproductive ray irritants or reproductive ray insufficiency in both sexes. Treatment of infertility caused by this disease, treatment of noveolar ovary/nymphomania syndrome in domestic animals, induction and enhancement of sexual behavior, or treatment of impotence and cold sensitivity. Reverse use (high dose effect) Contraception in females, suppression or delay of ovulation, induction of parturition, regulation of ovulation, suppression of oestrus, promotion of growth in female animals, secretion of progesterone, induction of menstruation, in the first three months of pregnancy. treatment of endometriosis, treatment of mammalian tumors and botanical tumors, treatment of polycystic ovary syndrome, (Stein-Leventhal) treatment of uterine cancer, benign prostatic hypertrophy and prostate cancer. treatment of male carnivores, contraception in males, treatment of diseases caused by excessive production of reproductive hormones in both sexes, functional castration of male carnivores, suppression of secretion during proestrus. Furthermore, these compounds and conventional LH−RH
The compound exhibits surprising new utility as an apparent abortifacient that suppresses circulating levels of HCG and acts directly on the placenta. This placental effect suggests its utility against chorionic carcinoma. In another aspect, the invention provides specific uses (including uses not described above for LH-RH congeners) of the compounds described above, namely, ovulation suppression (i.e., contraception) in females, uterine It relates to the treatment of endometriosis, the treatment of benign prostatic hypertrophy, and the inhibition of spermatogenesis in males (ie, contraception). That is, the present invention provides methods for suppressing ovulation, treating endometriosis, reducing prostate size, or spermatozoa by administering to a mammal an effective amount of the compound according to the present invention or a pharmaceutical composition containing the same as described above. This invention relates to a method of inhibiting formation. In practicing the methods of the invention, an effective amount of a compound of the invention or a pharmaceutical composition containing the same is administered to an individual in need or desire of treatment. These compounds or compositions can be administered by various routes depending on the end use, such as orally, parenterally (subcutaneously, intramuscularly and intravenously), intravaginally (particularly for contraceptive use), intraanally, Can be administered intrabuccally (including sublingually) or intranasally. The most appropriate route of administration will depend on the use, the type of active ingredient, the recipient animal, and the judgment of the physician. The compounds or compositions of the invention can also be administered by slow release, deposit or fill formulations, as detailed below. Generally, for the above-mentioned applications, referred to as "reverse utilization" or high dose utilization, the active ingredient may be administered at about 0.01 to 100 μg, preferably about 0.1 to 5.0 μg per kilogram of body weight per day. This administration can be administered once a day, several times a day or, for more effective results, by slow release. The precise dosage and method of administration of these compounds and compositions will depend on the individual being treated, the type of treatment,
It will depend on the extent of the disease and, of course, on the judgment of the doctor. Generally, for parenteral administration,
Lower dosages are required compared to other methods of administration that rely heavily on absorption. The compounds according to the invention are well tolerated and relatively non-toxic. When subcutaneously injecting representative compounds into mice, the LD ₅₀ is greater than 40 mg/Kg, which is very safe compared to the therapeutic doses mentioned above. In yet another aspect, the present invention provides a pharmaceutical composition comprising the compound according to the present invention as an active ingredient, which is formulated with a pharmaceutically acceptable non-toxic carrier. As mentioned above, such compositions may be administered parenterally (subcutaneously, intramuscularly or intravenously).
For administration, especially in the form of liquid solutions or suspensions, for intravaginal or anal administration, especially in semisolid forms such as creams and suppositories, for oral or buccal administration, especially in tablets or It can be applied in the form of capsules, in particular in the form of powders, nasal solutions or aerosols for intranasal administration, respectively. The compositions of the invention may be administered in any unit dosage form and may be administered, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing
Company, Easton, Pa., 1970, by any method well known in the pharmaceutical industry. Preparations for parenteral administration may include the usual excipients, sterile water or saline, polyethylene glycol and other polyalkylene glycols, vegetable oils, hydrogenated naphthalenes. Preparations for intravaginal or intraanal administration, such as suppositories, may include excipients such as polyalkylene glycols, petrolatum, cocoa butter, and the like. Preparations for administration by inhalation may be solid and may contain, for example, lactose as an excipient, and preparations in the form of nasal drops may be aqueous or oily solutions. Typical excipients used for buccal administration include sugar, calcium stearate, magnesium stearate, and pregelatinized starches. It is desirable that the compounds according to the invention be administered in a single manner over a long period of time, for example for one week to one year. A variety of slow release deposits or filled dosage forms are available. For example, pharmaceutically acceptable non-toxic salts of compounds with low solubility in body fluids can be included. Such salts include, for example (a) polybasic acids such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamonic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acid, polygalactronic acid, etc. Acid addition salts of acids; (b) zinc, calcium,
Salts of polyvalent metal cations such as bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc. or, for example, N, N′-
dibenzylethylenediamine or a salt of an organic cation formed from ethylenediamine; or (c)
Combinations of the above (a) and (b) include, for example, zinc tannate salt. Additionally, the compounds of the invention, or preferably relatively insoluble salts as described above, can be prepared in the form of a gel, such as an aluminum monostearate gel suitable for injection using sesame oil. Particularly preferred salts are zinc salts,
These include zinc tannate salt and pamonate salt. Other injectable slow release preparations include, for example, US Pat. No. 3,773,919
As described in , there are compounds or salts dispersed or encapsulated in slowly degrading, non-toxic, non-antigenic polymers such as polylactic acid/polyglycolic acid polymers. The compounds of the invention, or preferably relatively insoluble salts as described above, may also be formulated in the form of cholesterol matrix silastic pellets, particularly when administered to animals. Still other slow release deposits or filler formulations, such as intracellular fat particles, are well known and described in the literature. For example, “Sustained and Controlled
Release Drug Delivery Systems”JR
Robinson ed., Marcel Dekker, Inc., New
See York, (1979). A record of LH-RH type compounds can be found, for example, in US Pat. No. 4,010,125. Polypeptides according to the invention can be synthesized by techniques known in the polypeptide art. The technique used is JM
Stewart and JD Young, "Solid Phase Peptide Synthesis", W.
H. Freeman & Co., San Francisco (1969) and J. Meinfauer, Hormonal Proteins and Peptides, Vol. 2, P. 46, Academic Press (New York), 1973, (Solid Phase Peptide Synthesis);
E. Schroder and K. Lubke “The Peptide” No. 1
Volume, Academic Press (New York), 1965
(conventional solution synthesis). Generally, these methods consist of sequentially adding one or more amino acids or appropriately protected amino acids to a growing polypeptide chain.
Usually, either the amino group or the carboxyl group of the first amino acid is protected with a suitable protecting group.
The protected or derivatized amino acid is then attached to an inert solid support or left in solution, and the next amino acid with an appropriately protected complementary (amino or carboxyl) group is added to form an amide bond. It is used to add it under the appropriate conditions. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added. After all desired amino acids have been attached in the appropriate order, residual protecting groups (and solid support) are removed either sequentially or simultaneously to yield the final polypeptide. As a simple variation of this general approach, two or more amino acids can be added to the growing chain at once. For example, a protected polypeptide can be coupled with an appropriately protected dipeptide under conditions that do not racemize the chiral center to form a pentapeptide (after removal of the protecting group). The compounds according to the invention can also be obtained by solid phase peptide synthesis. In this method, the α-amino acid functional group of the amino acid is protected with an acid or base functional group. Such protecting groups should be stable under peptide bond-forming conditions and at the same time be easily removable without disruption of the growing peptide chain or racemization of chiral centers contained therein. There must be. Suitable protecting groups include t -butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t -amyloxycarbonyl, isobornyloxycarbonyl, α,
α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o -nitrophenylsulfenyl, 2-cyano- t -butyloxycarbonyl,
Examples include 9-fluorenylmethyloxycarbonyl, among which t -butyloxycarbonyl (Boc) is particularly preferred. Particularly preferred side chain protecting groups include for arginine: nitro, p -toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc and adamantyloxycarbonyl; for tyrosine: benzyl, o -bromobenzyl Oxycarbonyl, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; for serine: benzyl, tetrahydropyranyl; for histidine: benzyl, p -toluenesulfonyl and 2,4-dinitrophenyl There is. The C-terminal amino acid is attached to a suitable solid support. Suitable solid supports useful in the above-described syntheses are materials that are inert to the reaction reagents and reaction conditions of the multistage condensation-deprotection reaction and insoluble in the medium used. Suitable solid supports include chloromethyl polystyrene-divinylbenzene polymer, hydroxymethyl-polystine-divinylbenzene polymer, and the like, with chloromethyl-polystyrene-1% divinylbenzene polymer being preferred. C-
In the special case where the terminal end is glycinamide, P.
Rivaille, et al, Helv.Chim.Acta., 54, 2772
(1971) is a particularly useful support. Attachment to chloromethyl polystyrene-divinylbenzene type resins can be achieved by using N-protected amino acids, especially Boc-amino acids such as cesium, tetramethylammonium, triethylammonium,
4,5-diazabicyclo[5,4,0]undecene-5 or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF)
in the form of a solution of the cesium salt in a solvent such as, in particular, the form of its cesium salt dissolved in DMF at high temperatures, e.g.
from about 12 to 48°C at a temperature of from 60°C to 60°C, preferably at about 50°C.
This is accomplished by reacting with the chloromethyl resin for a period of time, preferably about 24 hours. The attachment of N〓-Boc-amino acid to benzhydrylamine resin is
N,N'- in a solvent such as dichloromethane or DMF, preferably dichloromethane, at a temperature of about 10 to 50°C, preferably 25°C, for about 2 to 24 hours.
Dicyclohexylcarbodiimide (DCC)/1
-Achieved by hydroxybenzotriazole (HBT) mediated coupling.
Subsequent coupling of protected amino acids can be performed using automated polypeptide synthesizers well known in the art. Removal of the N-protecting group is carried out, for example, in the presence of a solution of trifluoroacetic acid in methylene chloride, hydrogen chloride in dioxane, hydrogen chloride in acetic acid or other strong acid solution, preferably a 50% solution of trifluoroacetic acid in dichloromethane. Can be achieved at room temperature. Each protected amino acid is preferably used in about 2.5 molar excess and the coupling can be carried out in dichloromethane, dichloromethane/DMF mixtures, DMF, etc., preferably in methylene chloride at about room temperature. The coupling agent is usually a solution of DCC in dichloromethane, but also N,N'-di- iso -propylcarbodiimide or other carbodiimides alone or HBT, N-hydroxysuccinimide, other N-hydroxyimides or oximes. It may be used in the presence of Alternatively, protected amino acid active esters (eg p-nitrophenyl, pentafluorophenyl, etc.) or symmetrical anhydrides can be used. In the final step of solid phase synthesis, the fully protected polypeptide is removed from the resin. When the bond to the resin support is of the benzyl ester type, at a temperature of about 10 to 50°C, preferably about 25°C, about 12
for 24 hours, preferably about 18 hours, using an alkylamine or fluoroalkylamine for polypeptides with a proline C-terminus and e.g. ammonia/methanol or ammonia/ethanol for peptides with a glycine C-terminus. can be cleaved by aminolysis using . Alternatively, the polypeptide can be removed from the resin by transesterification, for example with methanol, and then aminolysis. The protected polypeptide can now be purified by silica gel chromatography. Removal of side chain protecting groups from polypeptides is carried out at a temperature of about -10 to +10°C, preferably about 0°C.
hydrogen fluoride/pyridine by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or other carbonium scavenger for about 15 minutes to 1 hour, preferably about 30 minutes, at by treatment with complexes, by treatment with tris(trifluoroacetyl)boric acid and trifluoroacetic acid, by reduction with palladium and hydrogen supported on carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia solution. , preferably by reduction with liquid hydrogen fluoride and anisole. In the case of glycine-terminated peptides attached to benzhydrylamine resins, the resin cleavage and deprotection steps can be accomplished in a single step using liquid hydrogen fluoride and anisole as described above. Completely deprotected polypeptides are then produced by a series of chromatographic techniques including some or all of the following techniques. Ion exchange on weak base resins in acetate form; underivatized polystyrene-divinylbenzene (e.g. Amberlite)
Hydrophobic adsorption chromatography using XAD); silica gel adsorption chromatography; ion exchange chromatography using carboxymethyl cellulose; partition chromatography or alternating current partition using e.g. Cephadex G-25; high performance liquid chromatography HPLC, especially reversed phase HPLC using octyl- or octadecylsilyl-silica bonded phase column packings. If a racemic amino acid is used at position 6, the diastereomeric nonapeptide or decapeptide is ultimately separated, and the desired peptide containing the D -amino acid at position 6 is preferably separated and purified by the chromatographic process described above. Ru. The preparation of peptides with a C-terminal azaglycinamide is preferably carried out according to conventional peptide solution synthesis methods using known peptide intermediates.
This method is detailed in Example 3. The present invention utilizes the formula () (pyro)Glu-His-Trp-Ser-Tyr-3-(2-
naphthyl)-D-alanyl-Leu-Arg-Pro-
The present invention relates to a method for producing a compound represented by Gly-NH ₂ ( ) and a pharmaceutically acceptable salt thereof. This method uses the formula () (pyro)Glu−His−Trp−Ser−Tyr−3−(2−
naphthyl)-D-alanyl-Leu-Arg-Pro-
A peptide fragment that can constitute a compound represented by Gly-NH ₂ () is coupled by peptide synthesis means, or a protecting group is removed from a protected compound of a polypeptide compound of formula () to produce a compound of formula () or and, optionally, (ii) converting the compound of formula () into a pharmaceutically acceptable salt; (iii) converting the salt of the compound of formula () into a pharmaceutically acceptable salt; or (iv) characterized in that the salt of the compound of formula () is converted into a free polypeptide compound of formula (); In order to facilitate understanding and implementation of the present invention,
This will be explained below using an example. These examples are not intended to limit the scope of the invention, but are merely representative. Preparation Example A 0.1 of anhydrous ethanol distilled using magnesium ethoxide was placed in a flask to be dried in an oven, and 1.52 g of sodium metal was added thereto. When hydrogen evolution ceased, 10.21 g of ethyl 2-acetamido-2-cyanoacetate and 13.26 g of 2-bromomethylnaphthalene were added to the solution. The solution was heated to reflux for 1 hour, then cooled. The ethanol was removed under reduced pressure and the residue was taken up with ethyl acetate. The organic layer was diluted with 50 ml of saturated sodium chloride solution twice with 50 ml of water.
ml and dried over magnesium sulfate. The solution was filtered, the solvent was removed under reduced pressure, and the residue was further hydrolyzed in 100 ml of concentrated hydrochloric acid under reflux for 2 hours. The hydrolysis mixture was cooled and the crude product precipitate was filtered off. The crude product was dissolved in concentrated hydrochloric acid treated with activated carbon.
The pH of the solution was adjusted to 6 using concentrated ammonium hydroxide. The precipitate was filtered and dried under reduced pressure to obtain pure 3-(2-naphthyl).
- 11.3 g of D , L -alanine was obtained. Melting point 230−232
℃. In place of 2-bromomethylnaphthalene, equivalent stoichiometric amounts of 1-bromomethylnaphthalene, 9-bromomethylanthracene, 9-bromomethylfluorene, 2-bromomethylfluorene, 2-bromomethylanthracene, 1-bromomethylanthracene, α-chloroisodyurene, 4-bromomethylbiphenyl, 1-bromomethyladamantane, 3-bromomethylphenanthrene, 1-chloromethyl-2,4,6-tri-(n-
Butyl)benzene and 1-chloromethyl-2,3,4,5,6-pentamethylbenzene were used to obtain the following amino acids by repeating the above procedure. 3-(1-naphthyl) -D , L -alanine, mp185-187℃, 3-(9-anthryl) -D , L -alanine, mp290℃ (HCl salt), 3-(9-fluorenyl) -D , L -alanine, 3-(2-fluorenyl) -D , L -alanine, mp264-269℃, 3-(2-anthryl) -D , L -alanine, 3-(1-anthryl) -D , L -alanine , 3-(2,4,6-trimethylphenyl) -D ,
L-alanine, mp235-237℃, 3-(4-biphenylyl) -D , L -alanine, mp290℃, 3-(1-adamantyl) -D , L -alanine, 3-(3-phenanthryl) -D , L -alanine, 3-(2,4,6-tri( n -butyl)phenyl) -D , L -alanine and 3-(2,3,4,5,6-pentamethylphenyl)D,L- Alanine. Preparation example B 1,1-diphenylethylene 18.2g, methyl
25.3g α-methoxy-N-benzyloxycarbonylglycinate and 2-naphthalenesulfonic acid
A solution of 1.5 g dissolved in 300 ml of dry benzene was refluxed for 2 days. The crude product is CH ₂ Cl ₂ −CH ₂ Cl ₂ /
Purification was performed in a silicic acid column using a density gradient of EtOAc (18:1). Purified methyl 2-[1-
(2,2-diphenylethylenyl)]-N-benzyloxycarbonylglycinate, KOH10.9g
Hydrolysis was carried out using 350 ml of a 10% aqueous methanol solution to obtain the corresponding acid. The obtained crude acid is concentrated
3-(2,2-diphenylmethyl) was dissolved in 100 ml of 95% ethanol containing 3 ml of HCl and hydrogenated for 24 hours in the presence of 2 g of 10% Pd supported on carbon.
- 2.4 g of D , L -alanine was obtained. mp235−237
℃. Preparation Example C 3-(2-naphthyl) -D , L -alanine 12.9g
6.23 ml of acetic anhydride and 60 ml of 1 M NaOH were added over 30 minutes to a solution of 120 ml of 1 M NaOH at 0°C. The pH was adjusted to 2 using concentrated hydrochloric acid, and the resulting precipitate was filtered off. The solid content was recrystallized from a 60% aqueous ethanol solution to obtain 12.2 g of N-acetyl-3-(2-naphthyl) -D , L -alanine. Add 15.8 ml of boron trifluoride etherate to a solution of 15 g of this N-acetylamino acid in 240 ml of dry methanol.
was added and the mixture was refluxed for 1 hour. The alcohol was evaporated, 200 ml of water was added and the solution was extracted with ethyl acetate. The organic layer was washed with an aqueous base solution and an acid,
Dry over MgSO ₄ , filter and evaporate volatiles to give an oil. This oil was crystallized from ethyl acetate/hexane to give methyl N-acetyl-3-
14.2 g of (2-naphthyl) -D , L -alaninate was obtained. mp79−80℃. 3-(2-naphthyl) -D , L -alanine is replaced by an equal stoichiometric amount of 3-(1-naphthyl) -D , L -alanine, 3-(2-flerenyl) -D , L -alanine, Repeat the above procedure using 3-(2-anthryl) -D , L -alanine, 3-(1-anthryl) -D , L -alanine and 3-(2,2-diphenylmethyl) -D , L -alanine The following compound was obtained. Methyl N-acetyl-3-(1-naphthyl)-
D, L -alaninate, mp97.5-98℃, Methyl/N-acetyl-3-(2-fluorenyl) -D , L -alaninate, mp170-171℃, Methyl/N-acetyl-3-(2-anthryl) )
-D , L -alaninate and methyl N-acetyl-3-(2,2-diphenylmethyl) -D , L -alaninate, mp113-
114℃. Preparation Example D Methyl N-acetyl-3-(2-naphthyl)-
6.6 g of D, L -alaninate was dissolved in a mixture of 300 ml of dimethyl sulfoxide, 120 ml of 1 M KCl and 780 ml of water, and the solution was mixed with 33.6 ml of the enzyme subtilisin.
Treated with a solution dissolved in 3 ml of 0.1 M KCl. The pH was maintained at 7 by automatic titration of 0.2 M NaOH using a Radiometer pH controller. Hydrolysis was stopped after 30 minutes when 70 ml of NaOH solution was consumed. in solution
12 g of NaHCO ₃ was added and extracted with ethyl acetate. The organic layer is methyl/N-acetyl-3-(2-naphthyl)
- Contained D -alaninate. Crystallization from methyl acetate/hexane gave a yellow solid.
mp80−81℃. The above compound was converted into free amino acids and further into N-Boc amino acids as follows. Methyl N-acetyl-3-(2-naphthyl)-
A solution of 2.5 g of D-alaninate in 60 ml of 6 N HCl
Heated to -130°C for 3 hours and cooled to room temperature. Collect the formed white precipitate and add 50 ml of water containing 1 ml of 12 N HCl.
was recrystallized by adding NH ₄ OH to adjust the pH to 6, dried under vacuum, and obtained 3-(2-naphthyl) -D.
- Obtained 1.2 g of alanine. mp242−244℃, [α]
25D26.6° (C 0.5, CH ₃ CO ₂ H). 3-(2-naphthyl) -D -alanine at 1N
It was dissolved in a mixture of 55 ml of NaOH, 10 ml of H ₂ O and 20 ml of dioxane, and 1.48 g of di- tert -butyl dicarbonate and 0.22 g of magnesium oxide were added to the solution at 0° C. while stirring. After 1.5 hours, an additional 0.3 g of di- tert -butyl dicarbonate was added and the mixture was allowed to cool to room temperature. Filter the solid content,
The filtrate was concentrated to 50ml. This aqueous solution
The pH was adjusted to 2.5 with NaHSO ₄ and extracted with ethyl acetate.
The organic layer was washed with 5% NaHSO ₄ , water, and then saturated brine. The ethyl acetate extract was dried over magnesium sulfate, filtered, and concentrated to an oil.
This oil was crystallized from ether/hexane to give N-Boc-3-(2-naphthyl) -D -alanine.
Obtained 1.3g. mp90−91°, [α]25D−32.6°(C
0.8, MeOH). Methyl N-acetyl-3-(2-naphthyl)-
Equistoichiometric amount of methyl N-acetyl-3-(1-naphthyl)- in place of D, L -alaninate
D, L -alaninate, Methyl N-acetyl-3-(2-fluorenyl)- D , L -alaninate Methyl N-acetyl-3-(2-anthryl)
- Repeat the above procedure using D , L -alaninate and methyl N-acetyl-3-(2,2-diphenylmethyl) -D , L -alaninate, respectively, and then proceed to the next N〓- via the corresponding free amino acid. Boc amino acid was obtained. N-Boc-3-(1-naphthyl) -D -alanine, mp92-93℃, [α]25D54.8°(C
0.5MeOH), N-Boc-3-(2-fluorenyl) -D -alanine, mp161-163°C. N-Boc-3-(2-anthryl) -D -alanine, and N-Boc-3-(2,2-diphenylmethyl)-
D-alanine, mp153-154°C. Preparation Example E 3-(2-naphthyl)- in a hydrogenation bottle
0.85 g of D-alanine, 100 ml of 2M hydrochloric acid, and 0.85 g of Adams catalyst (Pto ₂ ) were added. The solution was treated with 60 lb/ ⁱⁿ² _H2 gas for 20 hours in a Parr hydrogenation bottle. Heat the mixture to dissolve the white precipitate;
It was filtered using diatomaceous earth. The solution was concentrated under reduced pressure and lyophilized using water to obtain 0.8 g of 3-(2-perhydronaphthyl) -D -alanine. White solid, mp230−232℃. This material was dissolved in a mixture of 3.2 ml 1N NaOH, 5 ml water and 15 ml dioxane and treated with 0.14 g MgO and 0.85 g di- tert -butyl dicarbonate. After 1 h at 0 °C and 2 h at 25 °C, the suspension was filtered, evaporated to dryness under reduced pressure, the residue was dissolved in water, washed with diethyl ether,
_NaHSO4 was added to bring the pH to 2. This acidified aqueous layer was extracted three times with ethyl acetate, the extracts were collected,
Dry over _MgSO4 , filter and concentrate to a white oil. N-Boc-3-(2-perhydronaphthyl)-
0.75 g of D-alanine was obtained. A portion (0.1 g) of the above material was dissolved in 5 ml of tetrahydrofuran and titrated with freshly prepared diazomethane at 0°C until completely bright yellow. The reaction mixture was cooled with 1 ml of acetic acid, the solvent was evaporated and the residue was partitioned between 75 ml of ethyl acetate and 75 ml of water. The organic layer was dissolved in 5% NaHCO ₃ , water, 5% NaHSO ₄ ,
Washed with water and saturated NaCl solution and dried over _MgSO4 . Filter the solution and concentrate under reduced pressure, filter the solution and concentrate under reduced pressure on a preparative thin layer chromatography plate (750 μ thick, silica gel, 20 × 20 cm)
loaded into. The chromatography plate was developed with dichloromethane/ethyl acetate (18/1) to remove the product band. The silica gel from the product band was washed with dichloromethane/ethyl acetate (9:1) on a glass filter and the filtrate was concentrated to give a light yellow oil of methyl N-Boc-3-(2-perhydronaphthyl) -D- 0.1 g of alaninate was obtained. This material was obtained as a mixture of two isomers at the 2 positions of the perhydronaphrene nucleus. These diastereomeric compounds were separated using a high performance liquid chromatography column (Lichrosorb (trade name) silica gel 60, 5 microns) using ethyl acetate/hexane (1:9) as the eluent, and hydrolyzed. Free acid N-Boc-3
-(2-perhydronaphthyl) -D -alanine was obtained. 3-(2-naphthyl) -D -alanine was replaced with equivalent stoichiometric amounts of 3-(1-naphthyl)-D-alanine, 3-(2,2-diphenylmethyl) -D -alanine, 3-(2-naphthyl) -D -alanine, 3-(2-naphthyl)-D-alanine, ,4,6-trimethylphenyl) -D ,
L-alanine, 3-(4-biphenylyl) -D , L -alanine, 3-(2,4,6-tri(n-butyl)phenyl) -D - L -alanine and 3-(2,3,4 , 5,6-pentamethylphenyl) -D , L -alanine were used to obtain the next N-Boc amino acid. N-Boc-3-(1-perhydronaphthyl) -D
-Alanine, N-Boc-3-(perhydro-2,2-diphenylmethyl) -D -alanine, N-Boc-3-(2,4,6-trimethylcyclohexyl) -D , L -alanine, N-Boc- 3-(perhydro-4-biphenylyl) -D , L -alanine, N-Boc-3-(2,4,6-tri( n -butyl)cyclohexyl) -D , L -alanine and N-Boc-3- (2,3,4,5,6-pentamethylcyclohexyl) -D , L -alanine. Example 1 (Reference Example) Benzhydrylamino-polystyrene-divinylbenzene resin (Love Systems, Inc.) as described above for Rivalle in a Beckman 990 polypeptide synthesis reactor.
0.8g (0.8mmol) was charged. Subsequently, amino acids were added to this resin according to the following synthetic procedure. Step 1 CH ₂ Cl ₂ washing 1.5 minutes once 2 50% CF ₃ CO ₂ H/CH ₂ Cl ₂ … Deprotection group 1.5 minutes once 3 50% CF ₃ CO ₂ H/CH ₂ Cl ₂ … Deprotection group 1 3 times 30 minutes 4 CH ₂ Cl ₂ washing 3 times 1.5 minutes 5 10% triethylamine/CH ₂ Cl ₂ 2 times 1.5 minutes 6 CH ₂ Cl ₂ washing 3 times 1.5 minutes 7 N-Boc-amino acid solution Added once 8 N , N'-dicyclohexylcarbodiimide solution Add once 9 CH ₂ Cl ₂ Rinse and hold… Coupling
Coupling reaction time 2 hours 10 CH ₂ Cl ₂ ... Rinse 1 time 1.5 minutes 11 CH ₂ Cl ₂ washing 3 times 1.5 minutes 12 Ethanol washing 3 times 1.5 minutes 13 CH ₂ Cl ₂ washing 3 times 1.5 minutes To step 1-13 Thus, the coupling cycle for one amino acid is completed. Completion of the reaction is E.
Verified by the ninhydrin method of Kaiser et al., Anal.Biochem. , 34 , 595 (1970). The resin was subsequently coupled with a 2.5 molar excess of each protected amino acid and DCC. That is, the resin was treated with the following compounds during successive coupling cycles. 0.433g Boc-Gly-OH, 0.432g Boc-PrO-OH, 0.857g Boc-Arg(tosyl)-OH, 0.462g Boc-Leu-OH, 0.504g Boc-3-(2-naphthyl) -D -alanine and 0.272g 1-hydroxybenzotriazole 0.724g N-Boc-O-2-bromobenzoyloxycarbonyl-L-tyrosine, 0.59g Boc-Ser(benzyl)-OH, 0.608g Boc-Trp-OH, 0.654g Boc-His (tosyl)-OH and 0.524 g pyroglutamic acid The resin was removed from the reaction vessel, washed with CH ₂ Cl ₂ and vacuum dried to remove the protected polypeptide resin.
Obtained 2.0g. The polypeptide product was simultaneously removed from the resin and treated with anhydrous liquid HF to completely remove the protecting groups. A mixture of 2.0 g of protected polypeptide resin and 2 ml of anisole (delaxation agent) was placed in a Kelu F reactor and anhydrous liquid HF20 redistilled using _CoF3 .
ml for 30 minutes at 0°C. The HF was evaporated under reduced pressure to give (pyro)-Glu-His-Trp-Ser-Tyr.
-3-(2-naphthyl)- D -alanyl-Leu-
Arg-Pro-Gly- _NH2 (HF salt) was washed with ether. The residue was then extracted with glacial acetic acid. The acetic acid extract was freeze-dried to obtain 0.8 g of crude product. The crude polypeptide was treated with a 4 x 40 cm Amberlite (trade name) XAD-4 column (polystyrene-4% divinylbenzene copolymer) and water (0.5%).
It was eluted using a concave gradient from to ethanol (1). Tubes containing fractions of 1.470 ml from 690 ml of effluent solution were collected and stripped to dryness, yielding 490 ml of moderately purified polypeptide. Sephadex (trade name) G-25
Column (3 x 50 cm) and 1-butanol/toluene/acetic acid/water containing 1.5% pyridine (10:15:12:
150 ml of the above moderately purified product was subjected to partition chromatography using a solvent system consisting of 18). Pure fractions were subjected to thin layer chromatography (silica gel, BuOH/H ₂ O/HOAc/EtOAc = 1:
1:1:1) and HPLC (5 micron, reverse phase, octadecylsilyl packing, 40% _0.03MNH4OH /
60% acetonitrile). The desired product was obtained from the column in fractions with an eluate volume of 1000 ml to 1400 ml (Rf 0.1). The pure fractions were collected, strip dried, extracted with water and lyophilized to give pure pyro-glutamyl-histidyl-tryptopyl-seryl-tyrosyl-3-(2-naphthyl) -D -alanyl-leucyl-arginyl-prolyl. -Glycinamide was obtained in the form of an acetic acid addition salt. Yield 57mg,
[α] ²⁵ _D −27.4° (C 0.9, HOAc), mp185−193°C
(Disassembly). Example 2 (Reference Example) A synthetic program identical to that described in Example 1 was used for the synthesis of a congener with a C _- terminal Pro-NH- _CH2CH3 . 2.13 g of Boc-Pro-O-resin obtained by equimolar reaction of dry cesium salt of Boc-Pro-OH with chloromethyl-polystyrene/1% divinylbenzene (Lab Systems, Inc.) in a Beckman 990 synthesis reaction vessel. was loaded.
The amount of Boc-Pro-O-resin collected is proline
It contained 1.4 mmol. resin, protected amino acids and
Coupling was carried out sequentially with a 2.5 molar excess of DCC. Thus, the resin undergoes continuous coupling cycles.
1.61g Boc-Arg(tosyl)-OH, 0.93g Boc-
Leu-OH _H2O , 0.94 g Boc-3-(2-naphthyl) -D -alanine and 0.49 g 1-oxybenzoriazole, 1.75 g N-Boc-O-2-bromobenzyloxycarbonyl- L -tyrosine,
and 1.11 g of Boc-Ser(benzyl)-OH. At this point in the synthesis, the protected polypeptide resin is divided into two halves and one half is
Successive reactions with 0.57 g Boc-Trp-OH, 0.77 g Boc-His (tosyl)-OH, and 0.21 g pyroglutamic acid led to completion. The resin was removed from the reaction vessel, washed with methylene chloride, and then dried in vacuo to yield 2.26 g of protected polypeptide resin. Add the protected polypeptide to 25 ml of ethylamine.
Aminolysis was performed at 2°C for 18 hours to cleave it from the resin. The ethylamine was evaporated and the resin was extracted with methanol. Evaporate the methanol
Pyro-Glu-His (Tosyl)-Trp-Ser (Benzyl)
-Tyr(2-bromobenzyloxycarbonyl)-
3-(2-naphthyl) -D -alanyl-Leu-Arg
1.39 g of (tosyl)-Pro-NH-CH ₂ CH ₃ was obtained. The crude polypeptide was deprotected by treatment with a mixture of 3 ml anisole and 30 ml redistilled (from CoF ₃ ) anhydrous liquid hydrogen fluoride for 30 minutes at 0° C. in a Kel-F reaction vessel. Hydrogen fluoride was evaporated under vacuum and the residue was washed with ether.
The residue was dissolved in 2M acetic acid and lyophilized to give the crude (pyro)-Glu-His-Trp- as its acetate salt.
Ser-Tyr-3-(2-naphthyl) D -alanine-
0.82 _g of Leu-Arg-Pro-NH- _CH2CH3 was obtained. 0.9 x 550 filled with 40-50μ octadecylsilsylate silica (Merck, Lichroprep _C18 )
Final purification was performed using a preparative high-precision liquid chromatography operation on 20 ml of the sample using a mm column. Eluent: 0.03M NH ₄ OAc64%/acetonitrile
It was 36%. Total amount of crude material 61g after 4 operations
was purified. After freeze-drying with water three times,
Pure pyroglutamyl-histidyl-tryptopyl-seryl-tyrosyl- as its acetic acid addition salt
3-(2-naphthyl) -D -alanyl-leucyl-
15 mg of arginyl-proline ethylamide was obtained.
Melting point 180-190°C, [α] ²⁵ _D −57.2° (1.1, HOAc). The above cleavage is repeated by replacing ethylamine with equistoichiometric amounts of n-butylamine, cyclopropylamine, cyclohexylamine, trifluoromethylamine, pentafluoroethylamine, and 2,2,2-trifluoroethylamine, and The corresponding n-butylamide, cyclopropylamide, cyclohexylamide, trifluoromethylamide, pentafluoromethylamide, and 2,2,2-trifluoroethylamide were obtained. Example 3 A compound of the formula where Z is [Formula] was prepared by typical solution synthesis. It could be obtained as a free peptide or a salt, for example, by the following procedure (in the figure, "AzaGlyNH ₂ " is [formula]). [Table] Coupling of individual fragments is performed using the acylazide method (J. Honzel, et al., Coll. Czech
−20 by Chem. Comm , 26 , p. 2333 (1971)
℃ to 4℃, for example, by using a 10% molar excess of the acyl azide component in DMF containing about 6 equivalents of HCl and 1.4 equivalents of t-butyl nitrite, or by the DCC/HBT coupling method. , 10% molar excess of N-terminal pentapeptide acid and H-3-(2-naphthyl)-D-
Ala-Leu-Arg-Pro-Gly- _NH2 or H-3
-(2-naphthyl)-D-Ala-Leu-Arg-Pro-
This is done by using azaGly-NH ₂ and also a 10% molar excess of coupling agent or by other non-racemized fragment coupling techniques. Compounds (1) and (2) are known {M. Fujino, et al.
Biochem.Biophys.Res.Comm., 57 , 1248 (1974)
and ASDutta, et al , J.Chem.Soc.Perkin
I, 1979 , 379}. Compound (3) is obtained from compound (2),
Removal of Cbz and hydrogenolysis of the nitro group followed by a 10% molar excess of N-BOC-3-(2- It was produced by coupling with (naphthyl)-D-alanine.
For similar LHRH analog synthesis, reference may be made to Dutta et al., supra. Alternatively, [3-(2-naphthyl)-D- ^alanyl6 , ^azaGly10 ]-LHRH is pGlu-His-Trp
-Ser-Tyr-Gly-Leu-Arg-Pro-OH, which is prepared by solid phase peptide synthesis on conventional Merrifield resin, as described above,
It can be prepared by coupling in a similar manner to 10% molar excess of semicarbazide hydrochloride ( _NH2 -NHO= _-C -NH2.HCl) and DCC/HBT. In both cases above, DMF is the preferred solvent. After purification by preparative high performance liquid chromatography as described in Example 1, the desired product is
[3-(2-naphthyl)-D- ^alanyl6 ]LHRH
([α] ²⁵ _D −27.4° [c0.9, HOAc]) and [3−(
2
-naphthyl)-D- ^alanyl6 , ^azaGly10 - _NH2 ]
LHRH ([α] ²⁵ _D −34.3° [c0.3, HOAc]) was obtained. Example 4 A (Pyro)Glu-His-Trp-Ser-Tyr-3
-(2-naphthyl)-D-Ala-Leu-Arg-Pro
-Gly-NH ₂ (see example 1) hydrogen fluoride salt 0.1g
Dowe
(trade name) was passed through a column filled with anion exchange resin. The column was eluted with deionized water and the effluent was lyophilized (pyro)Glu-His-
Trp-Ser-Tyr-3-(2-naphthyl) -D-
The corresponding acetate salt of Ala-Leu-Arg-Pro-Gly- _NH2 , [α] ^25D _- 27.5° (C0.9, HOAc) was obtained. The above procedure was repeated to replace acetic acid with another acid in the resin equilibrium to obtain the corresponding salts such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, benzoic acid, etc. Acid addition salts of other compounds of formula can be prepared similarly. B In the case of salts with low solubility in water, they could be prepared by precipitation from water using the preferred acid. For example: Zinc tannate salt - (pyro)Glu in 0.1 ml of water
-His-Trp-Ser-Tyr-3-(2-naphthyl)
- D -Ala-Leu-Arg-Pro-Gly-NH _A solution of 10 mg of acetate was treated with a solution of 8 mg of tannic acid in 0.08 ml of 0.25M NaOH. Immediately add to a solution of 5 mg of ZnSO ₄ heptahydrate dissolved in 0.1 ml of water.
A solution of LH-RH congener was added. The resulting suspension was diluted with 1 ml of water and centrifuged. The supernatant was decanted and the residue was washed twice with 1 ml of water by centrifuging the precipitate and decanting the supernatant. The precipitate was dried under reduced pressure and the previously named LH
15 mg of mixed zinc tannate salts of -RH congeners were obtained. Pamoate salt - ethanol 1.6ml and 0.25
50 mg of (pyro)Glu-His-Tyr-3-(2-naphthyl) dissolved in a mixture of 0.1 ml of M NaOH.
-D -Ala-Leu-Arg-Pro-Gly- _NH2 To the solution of acetate was added a solution of 11 mg of pamonic acid dissolved in 0.3 ml of 0.25 M HaOH. The solvent was removed under reduced pressure and the residue was suspended in 2 ml of water, centrifuged and the supernatant was decanted. the precipitate
Washed with 1.5 ml H ₂ O, centrifuged and the supernatant was decanted. The precipitate was dried under vacuum to form the pamoate of the LH-RH congener named above.
Obtained 54 mg. Other salts with low water solubility can be prepared in a similar manner. C. Preparation of acid addition salts from free peptides. (Pyro)Glu−His−Trp−Ser−Tyr− as the free base
3-(2-naphthyl)- D -Ala-Leu-Arg-
30 ml of IN acetic acid was added to a 50 mg solution of Pro-Gly- _NH2 . The resulting solution was freeze-dried to obtain 50 mg of the acetate salt of the LH-RH homolog named above. Similarly, other acid addition salts of formula () can be prepared by replacing acetic acid with another acid (in stoichiometric equivalents relative to the peptide), such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid. of salt was obtained. D Production of salts of metal cations, e.g. zinc salts
(Pyro)Glu-His dissolved in a mixture of 4 ml 0.25 M NaOH, 0.3 ml water, and 1 ml ethanol.
-Trp-Ser-Tyr-3-(2-naphthyl) -D
A solution of ZnSO ₄ heptahydrate dissolved in 0.2 ml of water was added to the -Ala-Leu-Arg-Pro-Gly-NH ₂ 50 mg solution. The precipitate was centrifuged and the supernatant was decanted. The precipitate was washed with 1 ml of water by centrifugation and decanting the supernatant. The precipitate was dried to yield 48 mg of the zinc salt of the LH-RH homolog named above. In a similar manner, salts of other polyvalent cations, such as calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., can be prepared. Example 5 (Pyro)Glu−His−Trp− dissolved in 25 ml of water
Ser-Tyr-3-(2-naphthyl)-D-Ala-Leu
−Arg−Pro−Gly−NH ₂ acetate 50 mg solution,
NaOH to obtain exchange ions (hydroxide ions)
50g filled with Dowexl (strong base, quaternary ammonium anion exchange resin) equilibrated with
passed through the column. The column was eluted with 150 ml of water and the eluate was lyophilized to yield 45 mg of the corresponding polypeptide as free base. Similarly, other acid addition salts of compounds of formula (), such as those mentioned in Example 4, may be converted to the corresponding free bases. Example 6 LH-RH congeners according to the invention are used as active ingredients, such as (pyro)Glu-His-Trp-Ser-Tyr-3-
(2-Naphthyl) -D -Alanyl-Leu-Arg-Pro
Typical pharmaceutical crude products containing -Gly- _NH2 itself or pharmaceutically acceptable salts such as acetic acid addition salts, zinc salts, zinc tannate salts, etc. are shown below. A Tablet for oral (e.g. sublingual) administration: 1 LH-RH congener 50.0 μg Comperssible Sugar,
USP 90.0mg Calcium stearate 4.0mg 2 LH-RH congener 30.0μg Compresible Sugar,
USP 98.5mg Magnesium stearate 1.5mg 3 LH-RH congener 25.0μg Mannitol, USP 88.5mg Magnesium stearate, USP 1.5mg Pre-gelatinized starch USP 10.0mg 4 LH-RH congener 200.0μg Lactose, USP 83.3 mg Pregelatinized Starch USP 15.0mg Magnesium Stearate, USP 1.5mg Manufacturing Method a Sufficient amount of water to form wet granules when LH-RH is mixed with the sugar portion of the excipient. dissolve in After thorough mixing, the granules are dried in a tray or fluid bed dryer. The dried granules are then sieved to break up large agglomerates. The granules are then compressed on a standard tablet machine to the specified tablet weight. b In this manufacture, all formulations contain 0.01% gelatin (USP). Gelatin is first dissolved in a water-soluble granulation solvent, followed by LH-RH
Treat congeners. The remaining steps are as in (a) above. Formulation 4 can also be used as a tablet for oral administration. B Long-acting intramuscular injectable preparation 1 Long-acting intramuscular injectable - Sesame oil gel LH-RH congener 1.0 mg Aluminum monostearate, USP 20.0 mg Add appropriate amount of sesame oil to make 1.0 ml Aluminum monostearate Combine with sesame oil and heat to 125°C under stirring until a clear yellow solution forms. This mixture is then sterilized under pressure for sterilization and then allowed to cool. The LH-RH homolog is then crushed and added aseptically. Particularly preferred LH-RH congeners are salts of low solubility, such as zinc salts, zinc tannate salts, permoates, and the like. These have a very long duration of action. 2 Long-acting intramuscular injectable - biologically degradable polymeric microcapsules LH-RH congener 1% 25/75 glycolide/lactide copolymer intrinsic viscosity number 0.5 99% The above formulation is combined with the following composition: Dextrose 5.0% CMC , Sodium salt 0.5% Benzyl alcohol 0.9% Tween80 (trade name) 0.1% Microcapsules (0-150μ) suspended at 100% by adding an appropriate amount of purified water
25 mg of the microcapsules are suspended in 1.0 ml of vehicle. C Aqueous solution for intramuscular injection LH-RH congener 25mg Gelatin, non-antigenic 5mg Add appropriate amount of water for injection to 100ml. Gelatin and LH-RH congeners are dissolved in water for injection and the filtrate is sterilized. D Aqueous solution for nasal administration LH-RH congener 250mg Dextrose 5gm Benzyl alcohol 0.9gm Add an appropriate amount of purified water to make 100ml. Dissolve the LH-RH homologue, dextrose, and benzyl alcohol in purified water, and add an appropriate amount of purified water to make 100 ml. E Suppository vehicle for rectal administration LH-RH congener 500 μg Witepsol H15 (trade name) 20.0 gm LH-RH congener is combined with molten Witepsol H15, mixed well and poured into 2 gm suppository molds. Example 7 Estrus suppression in rats Procedure: Female rats (Hilltop, Sprague Dawley,
Approximately 200 g (vagina open) was divided into five animals per cage, and each group was divided into two cages. Rats are injected subcutaneously twice a day for 14 days. The stage of the oestrus cycle is determined daily by vaginal contents application, and then body weights are recorded at 0.1 and 2 weeks.
Percentage of females showing partial oestrus suppression (i.e. only diestrus and proestrus but not estrus) from day 4 and females showing complete suppression of oestrus (i.e. only diestrus) from day 4 Record the percentage of The ED ₅₀ is derived from the straight line of best fit of the percent estrus suppression data. LH-RH congeners are administered as their acetate salts dissolved in saline containing 0.1% bovine serum albumin (BSA). The injection volume is 0.2 ml and the congener dose is 0.05, 0.1, 0.2 or 0.4 μg. Control (saline containing BSA) showed no estrus suppression. Combining partial and complete estrus suppression
_ED50 is as follows. Compound ED ⁵⁰ (μg/injection dose) (Pyro)Glu-His-Trp -Ser-Tyr-3-(2-naphthyl) -D -alanyl-Leu-Arg -Pro-Gly-NH ₂ 0.08 Example 8 Approximately 25 g each Six Swiss with a weight of
(Pyro)Glu−His−Trp in Webster mice
25 ml of an aqueous solution of -Ser-Tyr-3-(2-naphthyl) -D -alanyl-Leu-Arg-Pro-Gly- _NH2 (acetate) was injected subcutaneously. The dose was 40 mg/Kg or approximately 1 mg/mouse. No lethal effects were observed. The LD ₅₀ is therefore greater than 40 mg/Kg.

Claims (1)

[Claims] 1 (i) Formula (I) (pyro)G1u-His-Trp-Ser-Tyr-3-
(2-naphthyl)-D-alanyl-Leu-Arg-
A peptide fragment that can constitute a compound represented by Pro-G1y-NH ₂ () is coupled by peptide synthesis means, or a protecting group is removed from a protected compound of a polypeptide compound of formula () to obtain a compound of formula (). and, optionally, (ii) converting the compound of formula () into a pharmaceutically acceptable salt; and (iii) converting the salt of the compound of formula () into a pharmaceutically acceptable salt. , or (iv) a method for producing a compound represented by formula () and a pharmaceutically acceptable salt thereof, which comprises converting a salt of the compound of formula () into a free polypeptide compound of formula ().